Vascular basement membranes are composed of macromolecules such as collagen, laminin, heparan sulfate proteoglycans, fibronectin and entactin (Timpl, R., 1996, Curr Opin Cell Biol 8:618-24). Functionally, collagen promotes cell adhesion, migration, differentiation and growth (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27:93-127), and via these functions is presumed to play a crucial role in endothelial cell proliferation and behavior during angiogenesis, which is the process of formation of new blood vessels from pre-existing ones (Madri, J. A. et al., 1986, J. Histochem. Cytochem. 34:85-91; Folkman, J., 1972, Ann. Surg. 175:409-16). Angiogenesis is a complex process, and requires sprouting and migration of endothelial cells, proliferation of those cells, and their differentiation into tube-like structures and the production of a basement membrane matrix around the developing blood vessel. Additionally angiogenesis is a process critical for normal physiological events such as wound repair and endometrium remodeling (Folkman, J. et al., 1995, J. Biol. Chem. 267:10931-34). It is now well documented that angiogenesis is required for metastasis and growth of solid tumors beyond a few mm in size (Folkman, J., 1972, Ann. Surg. 175:409-16; Folkman, J., 1995, Nat. Med. 1:27-31). Several studies have shown that inhibitors of collagen metabolism have anti-angiogenic properties, supporting the notion that basement membrane collagen synthesis and deposition is crucial for blood vessel formation and survival (Maragoudakis, M. E. et al., 1994, Kidney Int. 43:147-50; Haralabopoulos, G. C. et al., 1994, Lab. Invest. 71:575-82). However, the precise role of collagen in basement membrane organization and angiogenesis is still not well understood.
The present invention relates to proteins comprising the NC1 domain of an alpha chain of Type IV collagen having anti-angiogenic properties. In particular, the present invention relates to the novel proteins Arresten, Canstatin and Tumstatin, and to biologically active (e.g., anti-angiogenic) fragments, mutants, analogs, homologs and derivatives thereof, as well as multimers (e.g., dimers) and fusion proteins (also referred to herein as chimeric proteins) thereof. These proteins all comprise the C-terminal fragment of the NC1 (non-collagenous 1) domain of Type IV collagen. More specifically, Arresten, Canstatin and Tumstatin are each a C-terminal fragment of the NC1 domain of the xcex11 chain, xcex12 chain and xcex13 chain, respectively, of Type IV collagen. In particular, Arresten, Canstatin and Tumstatin are monomeric proteins. All three arrest tumor growth in vivo, and also inhibit the formation of capillaries in several in vitro models, including the endothelial tube assay.
The present invention encompasses isolated and recombinantly-produced Arresten, also referred to herein as xe2x80x9cArrestin,xe2x80x9d which comprises the NC1 domain of the xcex11 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Arresten, multimers of the isolated Arresten and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins. Also encompassed are compositions comprising isolated Arresten, its anti-angiogenic fragments, or both, as biologically active components. In another embodiment, the invention features a method of treating a proliferative disease such as cancer, in a mammal where said disease is characterized by angiogenic activity, the method comprising administering to the mammal a composition containing anti-angiogenic Arresten or its fragments. The anti-angiogenic Arresten and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies to the isolated anti-angiogenic Arresten and its fragments.
The present invention also encompasses isolated and recombinantly produced Canstatin, which comprises the NC1 domain of the xcex12 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Canstatin, multimers of the isolated Canstatin and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins. Also encompassed are compositions comprising isolated Canstatin, its anti-angiogenic fragments, or both, as biologically active ingredients. In another embodiment, the invention features a method of treating a proliferative disease such as cancer, in a mammal, where said disease is characterized by angiogenic activity, the method comprising administering to the mammal a composition containing anti-angiogenic Canstatin or its fragments. The anti-angiogenic Canstatin and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies to the isolated anti-angiogenic Canstatin and its fragments.
The invention likewise encompasses isolated and recombinantly-produced Tumstatin, comprising the NC1 domain of the xcex13 chain of Type IV collagen, having anti-angiogenic activity, anti-angiogenic fragments of the isolated Tumstatin, multimers of the isolated Tumstatin and anti-angiogenic fragments, and polynucleotides encoding those anti-angiogenic proteins. Also encompassed are compositions comprising isolated Tumstatin, its anti-angiogenic fragments, or both, as biologically active ingredients. In another embodiment, the invention features a method of treating a proliferative disease such as cancer in a mammal, where said disease is characterized by angiogenic activity, the method comprising administering to the mammal a composition containing anti-angiogenic Tumstatin or its fragments. The anti-angiogenic Tumstatin and its fragments can also be used to prevent cell migration or endothelial cell proliferation. Also featured are antibodies to the isolated anti-angiogenic Tumstatin and its fragments.